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In this study we describe the isolation of Xcadherin-11, the Xenopus homologue to the mesenchymal cadherin-11. Similar to epithelial and neural cadherins, overexpression of Xcadherin-11 led to posteriorised phenotypes due to inhibition of convergent extension movement. Because zygotic expression of Xcadherin-11 starts with gastrulation, we analysed the ability of different growth factors involved in mesoderm differentiation to induce the expression of Xcadherin-11. Using the animal cap assay, we demonstrated that Xcadherin-11 is activated by Xwnt-8 or beta-catenin, but repressed by BMP-4. Activin did not induce Xcadherin-11 but its synergistic function was required for the Xwnt-8/beta-catenin-mediated activation of Xcadherin-11. Because Xcadherin-11 and Xenopus E- and N-cadherin are differentially regulated by growth factors in the Xenopus animal cap, our results also reveal that this assay provides a helpful model system to elucidate the molecular control mechanism of epithelial-mesenchymal conversion.
Fig. 3. Whole-mount in situ hybridisation of Xenopus albino embryos. (A,B) Stage-6 blastula showing slight positive staining of the animal hemisphere. (C,D) Stage-10.5 gastrula with positive staining of the animal hemisphere including the marginal zone. (E) Stage-23 embryo, cranial neural crest cells are stained. (F) Tailbud stage 28, staining was found in the mandibular crest segments surrounding the eye anlage and in the hyoidal and the branchial crest segments. (G) Transverse section through the head region of the stage-23 embryo shown in (E), mandibular crest cells lining the cement gland are labelled. (H) Transverse section through the head region of the stage-28 embryo shown in (F), mandibular crest cells (MCS) surrounding the optical vesicle are stained. Arrowhead, dorsal portion; arrow, caudoventral portion of MCS. (I) Transverse section through the trunk region of the stage-23 embryo shown in (E), staining is observed in the lateral mesoderm. (J) Transverse section through the trunk region of the stage-28 embryo shown in (F), staining is observed in the sclerotome and in both layers of the lateral mesoderm. (A,C,E,G–J) Anti-sense probe. (B,D) Sense probe. Arrow in (D) marks the blastopore lip. Dorsal side of the embryos shown in (E–J) are orientated to the left-hand side.
Fig. 5. Posteriorised phenotypes resulting from injection of Xcadherin-11 RNA at the dorsal side showed disorganised mesodermal and neural tissues. Left panel: embryos injected with 700 pg RNA, middle panel: embryos injected with 350 pg RNA, right panel: uninjected control embryo. (A,F,K) Embryos before paraffin embedding and sectioning. (B–O, except F,K) Transverse sections of embryos shown in (A,F,K). Transverse sections through the head region revealing amorphous brain structure (B,C,D), or eye anlage not separated from diencephalon (G, asterisk), fused somites (C, arrowhead), otic vesicle atypically thickened by neural tissue (D, arrow), missing notochord (C,D, arrowhead) or enlarged notochords (I,J). Transverse sections of the trunk region revealed very small neural tubes (E,J). Bar: 200 mm.
Fig. 6. Whole-mount immunostaining with N-CAM and Tor-70 antibodies revealed defects in neural tube and notochord formation after overexpression of Xcadherin-11. (A,B) Embryos stained with N-CAM antibody. (A) Uninjected control embryo. (B) Embryo injected with 700 pg Xcadherin-11 mRNA: in addition to severe defects in eye, brain and sucker formation, the embryo possesses a very small neural tube (arrow) which is localised more ventrally compared with the control, nerve fibres appeared normal (arrowhead). (C,D) Embryos stained with Tor-70 antibody. (C) Uninjected control embryo. (D) Embryo injected with 350 pg Xcadherin-11 mRNA: the notochord was elongated and kinked in the direction of the branchial arches (asterisk), additional Tor- 70-positive vesicles were formed along the notochord (arrows).
Fig. 7. Convergent extension movement was abolished in explants of the dorsal upper blastopore lips after overexpression of Xcadherin-11. (A,C,E) Explants directly after excision from the embryos. (B,D,F) Corresponding explants after 24 h of in vitro culture. (A,B) Explant of an uninjected control embryo. (C,D) Explant of a control embryo injected with 700 pg preprolactin mRNA. (E,F) Explant of an embryo receiving 700 pg Xcadherin-11 mRNA. Normal convergent extension of the control explants becomes obvious by a constriction and an elongation of the explant in the direction of the former blastopore.